The long-term goal of this project is to apply molecular approaches to characterize and manipulate Taxus metabolism for production of the anti-cancer agent paclitaxel (Taxol"), a potent anti-cancer agent approved by the FDA for the treatment of breast, ovarian, and lung cancers, as well as the AIDS-related Kaposi's sarcoma. This proposal extends our efforts to characterize genes identified during the previous funding period that are involved in the gene network controlling paclitaxel production by cultured cells. The overarching objectives of the proposed research are to study the function of genes that are differentially expressed in association with paclitaxel accumulation. The information collected will be used to suggest strategies for both stabilizing and increasing paclitaxel yields in cell culture as well as provide insight into regulation of plant secondary metabolism in general. The specific aims are 1) Functionally characterize key candidate genes from the prior funding period that are specifically implicated in the global regulation of paclitaxel biosynthesis, including two putative rate- influencing paclitaxel biosynthetic pathway genes, two potentially novel paclitaxel biosynthetic pathway genes, a master regulator of paclitaxel biosynthesis and a candidate global regulator of secondary metabolism. 2) Examine targeted gene expression in cell suspensions and specific subpopulations that accumulate higher levels of paclitaxel via RT-PCR to facilitate the uncovering of genes that regulate taxane biosynthesis in Taxus and establish a new approach to metabolic engineering by focusing on superior cell subpopulations in a heterogeneous culture. Sequence will be generated for 20,000 ESTs from normalized Taxus cuspidata cDNA libraries to provide an extensive new EST resource for the community and for use in the future design and construction of oligonucleotide arrays that will be applied in global expression profiling of both whole cultures and sorted cell populations. Identifying novel genes and regulators of paclitaxel accumulation, and secondary metabolism in general, will significantly advance commercial plant cell culture technology processes for supply of paclitaxel and other important plant-derived pharmaceuticals used to treat a variety of human ailments such as cancer and AIDS. Because production of paclitaxel in the U.S. and world depends on plant cell culture technology, this research will directly impact future paclitaxel supply for both current and emerging applications.